1. Field of the Invention
The invention relates to a method for inexpensive and rapid screening of resistance to avermectins and milbemycins in parasitic nematodes. The method generally relates to the diagnosis of resistance by analysis of defects in the membranes of the sensory neurons in the chemosensory apparatus (sensillum) of nematodes. The method specifically relates to the diagnosis of resistance by the use of lipophilic dyes that stain sensory neurons in the chemosensory apparatus (sensillum) in wild-type nematodes but do not stain the sensory nematodes or have a reduced level of staining in the resistant nematodes.
2. Description of the Background Information
A. The Nematode Caenorhabditis elegans
The free-living soil nematode Caenorhabditis elegans is a simple invertebrate animal which is small (adults of both sexes, hermaphrodites and males, are approximately 1 mm long) and easily cultured in the laboratory. Methods for growing C. elegans are well-known to those skilled in the art. Brenner, S., Genetics 77:71-94 (1974); "The Nematode Caenorhabditis elegans," W. B. Wood, ed., 1988, Cold Spring Harbor Laboratory. For example, C. elegans can be grown either on agar surfaces seeded with Escherichia coli bacteria as a food source or in liquid cultures containing E. coli. Under optimal conditions eggs develop into egg-laying adults in less than three days; unmated hermaphrodites produce approximately 300 progeny. Thus it is easy to produce large numbers of animals and assays utilizing nematodes can be performed rapidly and in small volumes. These advantages have been noted by others who have used C. elegans as a test organism for anthelmintic and nematocide evaluation. Ohba, K. et al., J. Pestic. Sci 9:91-96 (1984); Vanfleteren, J. R. et al., Nematologica 18:325 (1972); Platzer, E. G. et al., J. Nematol. 9:280 (1977); Simpkin, K. G. et al., J. Chem. Tech. Biotechnol. 31:66 (1981); Spence, A. M. et al., Can. J. Zool. 60:2616 (1982); Popham, J. D. et al., Environ. Res. 20:183 (1979).
Over the last 15 years, the biology of C. elegans has been the subject of an intense scientific research effort. As a result, this organism is now genetically and biologically the best understood metazoan species. Wood, supra; Kenyon, C., Science 240:1448-1453 (1988). Methods for the generation, isolation, and analysis of single gene mutations have been developed and are facilitated by the rapid growth and ease of culture noted above as well as by ability of hermaphrodites to reproduce by self-fertilization.
Many mutant nematode strains have been described which display specific, characteristic responses to different classes of chemical agents. For example, Brenner, S., Genetics 77:71-94 (1974), among others (review Rand, J. B. et al., Psychopharm. Bull. 21:623-630 (1985)), discloses a series of mutants of C. elegans which are resistant to the cholinesterase inhibitors aldicarb, lannate or trichlorfon. Rand et al., supra, note that mutants resistant to one of these compounds are resistant to all three.
Brenner, supra, as well as Lewis, J. A. et al., Neurosci. 5:967-989 (1980), and Lewis, J. A. et al., Genetics 95:905-928 (1980), disclose the construction of a series of mutants resistant to the anthelmintic levamisole. The authors identify three classes of mutants based upon phenotypic analysis. The authors state that the most resistant class of mutants might lack one class of pharmacologically functional acetylcholine receptors.
Tabuse, Y. et al., Carcinogenesis 4:783-786 (1983), discloses the construction of a set of nematode mutants which are resistant to the mammalian tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).
In addition to mutants directed against membrane localized events, many mutants with intracellular targets have been described. For example, Sanford, T. et at., J. Biol. Chem. 258:12804-12809 (1983), discloses the isolation of mutants which are resistant to the RNA polymerase II inhibitor, .alpha.-amanitin. The authors discuss the use of these mutants to identify structural genes encoding subunits of RNA polymerase or its effectors.
Nematodes have been shown to respond to a variety of bioactive compounds in the same chemical form as that to which the higher vertebrates respond. For example, Morgan, P. G. et al., Anesthesiology 62:738-744 (1985), discloses the identification of a series of mutants which respond characteristically to volatile anesthetics. The mutants are characterized through their phenotypic expression in response to induction of anesthesia.
Trent, C. et al., Genetics 104:619-647 (1983), discloses the construction of mutants that are defective in egg-laying. The authors define four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine.
To date, over 700 genes have been identified by mutations. Wood, supra, pp. 502-559. Stable strains carrying these mutations are easily maintained either on agar plates or, for long term storage, frozen in liquid nitrogen.
Thus, the nematode C. elegans provides a versatile, non-vertebrate, in vivo animal model of the higher eukaryotes, which is economical to maintain, technically simple to utilize, amenable to genetic manipulation, and inherently responsive to bioactive agents which modulate pharmacological and biochemical activities in the higher vertebrates and, especially, in man.
B. Ivermectin Resistance
The avermectins/milbemycins represent a class of broad spectrum anthelmintics that share cross-resistance. Related bacterial species produce these two classes of compounds. Hundreds of synthetic related compounds have been synthesized.
Avermectins are a relatively new class of anti-nematode and anti-arthropod compound with high potency and comparatively low mammalian toxicity. Avermectins are used extensively for the control of nematode parasites in animals, including humans. The commercially available avermectins are produced by minor chemical modifications of fermentation products isolated from soil bacteria of the genus Streptomyces. Currently, the most significant commercial product is 22,23 dihydroavermectin B.sub.1, also known as ivermectin and marketed as IVOMEC. Ivermectin can be obtained from country feed stores or veterinary supply houses.
Ivermectin, a member of the avermectin/milbemycin class of parasiticides, is a broad spectrum endectocide that has been safely used in several mammalian species including man. Over 60 countries have approved the use of this drug for controlling parasites in humans, cattle, sheep, horses, goats, pigs, dogs, or other mammals. (Campbell, W. C. et al., Science 221:823-828 (1983); Di Netta, J., in Ivermectin and Abamectin (Campbell, W. C., ed.), pp. 344-346, Springer-Verlag (1989).)
Other classes of broad spectrum anthelmintics have declined in usefulness as a result of the development of resistance by the parasites against which they have been used. The development of resistance to pesticidal and parasiticidal agents is a problem in the field. During the development of drug resistance, there is a change in the gene frequency of a population as a result of drug selection. Susceptible members of the population are removed by the toxic drug, while the resistant members then are allowed to expand into the parasitic population. Increased amounts of the drug are required to produce the same effect. However, organisms resistant to higher concentrations of the drug then consequently expand into the parasitic population. This is a problem to be expected when a drug is applied repeatedly and in high concentrations to a parasitic population.
The use level (the level at which the pesticide or parasiticide is applied) may differ from the actual level initially effective to kill the parasite. Consequently, resistance to low levels of the pesticide may have developed long before the resistance to the high levels is evident. The development of ivermectin resistance in the field is characterized by a failure to detect the early low-level development of resistance because ivermectin use is often repeated and intensive. The detection of resistance to ivermectin or other pestcontrolling agents while the proportion of resistant genotypes is still small, is essential to the control of resistance. Resistance to anti-nematode compounds (anthelmintics) in common use has been observed, generally too late for intervention. (Anthelmintic resistance in nematodes: extent, recent understanding, and future directions for control and research, Pritchard, R. K., Ent. J. Parisitol. 20:515-523 (1990).) Resistance to ivermectin, the most recently introduced and most potent anthelmintic, is an emerging problem and, accordingly, a diagnostic for ivermectin resistance is urgently needed.
The presence of an ivermectin-resistant organism was reported in South Africa 33 months after the drug was introduced and used on sheep. (Carmichael, I. et al., J. South African Vet. Assoc. 58:93 (1987).) In addition, there have been other field reports of ivermectin-resistant isolates of H. contortus from sheep in South Africa (van Wyk, J. A. et al., Vet. Rec. 123:226-228 (1988)), and reports of five other isolates suspected of resistance (van Wyk, J. A. et al., Onderstepoort J. Vet. Res. 56:41-49 (1989). In all of these reports, high use levels failed to produce efficiency of protection. Resistance to ivermectin was also reported in an H. contortus isolate from sheep in Brazil (Echevarria, F. A. M. et al., Vet. Rec. 124:147-148 (1989)). Four reports of ivermectin-resistant Otrifurcata from goats were reported from New Zealand (Watson, T. C. et al., N. Z. Vet. J. 38:50-53 (1990); Badger, S. B. et at., N. Z. Vet. J. 38:72-74 (1990); McKenna, P. B. et al., N. Z. Vet. J. 38:114, 117 (1990); Pomonoy, W. E. et al., N. Z. Vet. J. 40:76-78 (1992)). Ivermectin resistance was also discovered in the United States in a goat population infected with H. contortus (Craig, T. M. et at., Vet. Rec. 126:580 (1990)). Ivermectin resistance was also reported in Europe in a goat strain of Ostertagia spp. from Scotland (Jackson, F. et al., Res. Vet. Sci. 53:371-374 (1992); Jackson, F. et al., Vet Rec. 130:210-211 (1992)).
Tests for ivermectin resistance have required necroscopy or fecal egg count reduction (herein "FECR"). Further, the manufacturer's recommended use level has been employed as the threshold of resistance in these tests. The sensitivity of FECR tests for detecting ivermectin resistance at low frequencies is unknown. Reliance upon necroscopy following treatment is impractical as a routine procedure to monitor resistance. Accordingly, an in vitro diagnostic which is easily interpretable, inexpensive, and capable of detecting ivermectin resistance at frequencies below the manufacturer's recommended use level are needed. (Shoop, W. L., Parasitology Today 9:154-159 (1993)).
In vitro tests for detecting ivermectin resistance have been reported, although none of these tests are routinely used. All show resistance to use levels of ivermectin, and it is unclear whether low-level ivermectin resistance could be detected by these tests. The first test is a larval development test in vitro in which T. colubriformis eggs are incubated in the presence of various concentrations of ivermectin and the proportions of live larvae at various larval stages are compared (Giodano, D. J. et al., Vet. Parasitol. 30:139-148 (1988)). A second test is based on the in vitro development from the L.sub.1 to L.sub.3 larval stages of H. contortus (Taylor, M. A., Res. Vet. Sci. 49:198-202 (1990)). A third in vitro test assays development from the egg to the larval stage L.sub.3 of H. contortus (Lacy, E. et al., in Resistance of Parasites to Antiparasitic Drugs (Boray, J. C. et al., eds.), pp. 177-184, MSD AGVET Merck & Co. (1990); Hubert, J. et al., Vet. Rec. 130:442-446 (1992)). A fifth in vitro test examines larval motility of the L.sub.3 larval stage of H. contortus (Gill, G. H. et al., Int. J. Parasitol. 21:771-776 (1991)). Isoenzyme analysis has also been used to distinguish ivermectin resistant and susceptible strains (Echevarria, F. A. M. et al., Vet. Parasitol. 44:87-95 (1992)). Obviously, these types of tests are not amenable to rapid and inexpensive field testing.
Ivermectin resistance has been observed only in Trichostrongylus from the gastrointestinal tract of sheep and goats. However, the possibility of resistance in other worms has been raised. Ivermectin is currently used as a treatment for humans infected with O. volvulus. The main goal of this therapy is to eliminate the microfilariae. This use was suggested by the activity of ivermectin against microfilariae of D. immitis in dogs, O. cervicalis in horses, and O. gibsoni in cattle (Egerton, J. H. et al., Br. Vet. J. 136:88-97 (1980); Blair, L. S. et al., Am J. Vet. Res. 44:475-477 (1983); Klei, T. R., et al., J. Parasitol 66:859-861 (1980); Egerton, J. R. et al., Vet. Parasitol 8:83-88 (1981); Forsyth, K. P. et al., Exp. Parasitol 58::41-55 (1984)).
Ivermectin resistance has been analyzed in the model nematode C. elegans by isolating ivermectin-resistant mutants (Johnson, C. D. et al., Abstracts of Int. C. Elegans Meet., p. 189 (1989); Day, C. H. et al., Abstracts of Int. C. Elegans Meet., p. 62 (1989); Kim et al., Abstracts Int. C. Elegans Meet., p. 184 (1989)). The results of these analyses have shown the following. A single mutation confers low-level resistance (5 or 10 ng/ml) in one of 23 avr genes distributed over five chromosomes. High-level resistance (20-25,000 ng/ml) is conferred by single recessive mutations of defined avr genes, dominant mutations, or two mutations in new avr genes. Most of the resistance at high levels of ivermectin requires mutations in two genes. The vast majority of ivermectin-resistant mutants are resistant to low levels of ivermectin. The significance of these studies is that the prevalent resistance observed in parasites will probably be low-level resistance. Ivermectin resistance observed in parasitic nematodes (Shoop, W. L., supra) shows that the level of resistance is similar to that observed in low-level ivermectin-resistant strains of C. elegans. This result could be predicted from the C. elegans experiments discussed above.